Inhibition Of Neoangiogenesis Research Articles

Apatinib in recurrent or metastatic head and neck cancer patients.

e18010 Background: Apatinib is a small tyrosine kinase inhibitor targeting the vascular endogenous growth receptor 2 (VEGFR-2) that shows potent antitumor activities in various advanced cancers via the inhibition of neo-angiogenesis. The effect of apatinib in recurrent or metastatic head and neck cancers has not been fully demonstrated. Methods: Patients with recurrent or metastatic head and neck cancers consecutively treated in our institute by apatinib from January 2015 to December 2020 were enrolled. Daily 250 mg or 500 mg apatinib was given with or without chemotherapy according to patients' tolerance. A part of the patients also received palliative radiotherapy. Disease response, treatment-related toxicities and patient survival were analyzed. Toxicity was assessed by CTCAE v4.0. Kaplan-Meier analysis was used to estimate patients' overall survival. The R software (version 3.6.3) were used for statistical analysis. Results: A total of 73 patients were enrolled in this study. There were 59 males and 14 females with the median age of 55 (range: 27-80) years. Most patients had a performance status of 0 (15/73, 20.5%) and 1 (51/73, 69.9%). There were 29 oral cavity cancers, 20 nasopharyngeal carcinomas, 8 salivary gland cancers, 7 nasal and paranasal sinus cancers and 9 other malignancies. Pathologically, 47 were squamous cell carcinoma, 18 were adenoid cystic carcinoma, and the rest 8 were adenocarcinoma, sarcoma and melanoma. The majority of patients were with metastatic diseases (57/74, 78.1%). Nine of the 73 (12.3%) received apatinib as first-line treatment, 22 (30.1%) as second-line and 42 (57.5%) as more than second-line treatments. Forty patients received S-1 or capecitabine chemotherapy, 2 received nab-paclitaxel and 1 received gemcitabine. Ten patients received palliative radiotherapy. The most frequent toxicities were anemia (37/73, 50.7%), leucopenia (27/73, 37.0%), thrombocytopenia (20/73, 27.4%), proteinuria (20/73, 27.4%), arterial hypertension (17/73, 23.3%), hand-foot syndrome (17/73, 23.3%). Hemorrhage occurred in 10 (13.7%) patients. No treatment-associated death was noted. The median follow-up was 29.9 months. Fifty-six patients had evaluable tumor response. Complete response occurred in 1 patients. Partial response was seen in 3 patients. Most patients had stable disease (29/56, 51.8%) or progressive disease (23/56, 41.1%). By the end of the last follow-up, 33 patients died of disease progression. The median overall survival was 29.6 months. Conclusions: Apatinib showed modest antitumor activities in recurrent or metastatic head and neck cancers. The side effects were manageable. The effect of apatinib in recurrent or metastatic head and neck cancers warrants further studies in larger-scale randomized studies.

A Prospectivly Randomized Phase-II Trial of Axitinib versus Everolimus as Second-Line Therapy in Metastatic Renal Cell Carcinoma (BERAT Study)

Introduction: Inhibition of neo-angiogenesis is a cornerstone of medical treatment in metastatic renal cell carcinoma (mRCC). While 1st line therapies were previously dominated by inhibitors of the vascular endothelial growth factor axis, 2nd line options were less clearly defined. We investigated the role of everolimus (EVE) or a tyrosine kinase inhibitor (TKI) in 2nd line treatment of mRCC patients. Methods: Key inclusion criteria were measurable mRCC, ECOG 0–1, IMDC risk: good or intermediate and adequate organ function. Patients who progressed on or were intolerant to bevacizumab + interferon were subject for randomization between TKI and EVE treatment. Cross-over occurred at time of progression during 2nd line treatment. Improvement of 2nd line progression-free survival (PFS) rate (PFR) at 6 months from 50% to 65% was the primary endpoint. Secondary endpoints were PFS, total PFS, objective response rate (ORR), overall survival (OS), safety, and patient reported outcomes. Results: In 2012–2015, a total of 22 patients were included. The study was stopped for poor accrual. Ten patients (46%) were randomized to receive 2nd line treatment with EVE (n = 5) or axitinib (n = 4)/sunitinib (n = 1). ECOG 0 was recorded in 20% (EVE) and 60% (TKI). Severe adverse events occurred in approx. 60% in each arm. ORR was 1/5 (20%) for TKI and 0/5 (0%) for EVE. PFR at 6 months was 20% in each arm. Median PFS was 3.7 months (EVE) and 2.2 months (TKI) (hazard ratio [HR] 1.0 [95% confidence interval [CI]: 0.26–3.85]). The OS was comparable between arms HR 1.12 (95% CI: 0.27–4.61). Conclusion: The rapid change of the treatment landscape, the limited use of bevacizumab and interferon in 1st line and the duration of 1st line treatment jeopardized BERAT trial recruitment. The small number of patients is a major limitation of our trial. Our observation indicated the poor prognosis in progressive patients and the limited efficacy of TKI or mTOR inhibitors in 2nd line treatment.

An insight into cytotoxic activity of flavonoids and sesquiterpenoids from selected plants of Asteraceae species.

Cancer continues to be a disease that is difficult to cure and the current therapeutic regimen is associated with severe side effects and the issue of emerging drug resistance. According to the World Health Organization fact sheet 2017, cancer is the second major cause of morbidity and death and a 70% rise in new cases is expected over the next 20years. The quest for new anticancer chemical entities is a thrust area identified by many government agencies and industry research and development groups. Nature-derived entities have played a very important role in therapeutics especially cancer Asteraceae is a large family consisting of around 1700 genera and more than 24,000species. Several genera belonging to this family have ethnopharmacological uses such as cytotoxicity, antidiabetic, hepatoprotective and antioxidant. This review highlights the cytotoxic potential of structurally novel flavonoids and sesquiterpenes isolated from some selected species of Asteraceae plants native to Asia, Europe, parts of Africa and America. The existing literature suggests that sesquiterpenes and flavonoids from various species of Asteraceae represent a viable class of secondary metabolites with strong cytotoxic potential. These have demonstrated potent activity in cell cycle arrest, inhibition of neoangiogenesis and induction of apoptosis. The sesquiterpenoids exhibiting potent cytotoxic activity were found to contain an α- methylene-butyrolactone conjugated with an exomethylene group and the flavonoids obtained from various plant species of Blumea suggest that a dihydroxy ring system present in structure is essential for activity. Most of the published literature contains in vitro data of extracts/secondary metabolites with very few in vivo studies. Additionally, there is dearth of knowledge on mechanisms of cytotoxic activity and molecular targets impacted by the active secondary metabolites. This review hopes to fuel interest in researchers to take up detailed investigations on these scaffolds that could contribute significantly as potential leads in anticancer drug development.

The antitumoral effects of chemerin are independent from leukocyte recruitment and mediated by inhibition of neoangiogenesis.

Chemerin, a multifunctional protein acting through the receptor ChemR23/CMKLR1, is downregulated in various human tumors and was shown to display antitumoral properties in mouse models of cancer. In the present study, we report that bioactive chemerin expression by tumor cells delays the growth of B16 melanoma and Lewis lung carcinoma in vivo. A similar delay is observed when chemerin is not expressed by tumor cells but by keratinocytes of the host mice. The protective effect of chemerin is mediated by CMKLR1 and appears unrelated to the recruitment of leukocyte populations. Rather, tumors grown in the presence of chemerin display a much smaller number of blood vessels, hypoxic regions early in their development, and larger necrotic areas. These observations likely explain the slower growth of the tumors. The anti-angiogenic effects of chemerin were confirmed in a bead sprouting assay using human umbilical vein endothelial cells. These results suggest that CMKLR1 agonists might constitute therapeutic molecules inhibiting the neoangiogenesis process in solid tumors.

Involvement of Alarmins in the Pathogenesis and Progression of Multiple Myeloma.

Objective: Multiple Myeloma (MM) is a haematological disease resulting from the neoplastic transformation of plasma cells. The uncontrolled growth of plasma cells in the bone marrow and the delivery of several cytokines causes bone erosion that often does not regress, even in the event of disease remission. MM is characterised by a multi-step evolutionary path, which starts with an early asymptomatic stage defined as monoclonal gammopathy of undetermined significance (MGUS) evolving to overt disease. Data Sources and Study Selection: We have selected scientific publications on the specific topics “alarmis, MGUS, and MM”, drawing from PubMed. The keywords we used were alarmines, MGUS, MM, and immune system. Results: The analysis confirms the pivotal role of molecules such as high-mobility group box-1, heat shock proteins, and S100 proteins in the induction of neoangiogenesis, which represents a milestone in the negative evolution of MM as well as other haematological and non-haematological tumours. Conclusions: Modulation of the host immune system and the inhibition of neoangiogenesis may represent the therapeutic target for the treatment of MM that is capable of promoting better survival and reducing the risk of RRMM.

Lenvatinib-related renal microangiopathy: a case series.

Tyrosine kinase inhibitors play an important role in the armamentarium against cancer. Lenvatinib is a multiple kinase inhibitor approved by the Food and Drugs Administration (FDA) for the treatment of advanced and radioresistant thyroid carcinomas and, in combination with everolimus, for renal cell carcinoma and unresectable hepatocellular carcinoma. The anti-tumoral activity is largely dependent on inhibition of neo-angiogenesis, and established side effects of anti-angiogenetic therapeutics include renal thrombotic microangiopathy (TMA). Here, we describe three cases of biopsy-proven renal TMA clinically presenting with proteinuria and stable serum creatinine in patients receiving lenvatinib for thyroid cancer. Microangiopathic lesions included glomerular basement membrane reduplication with segmental cellular interposition, mesangiolysis, and focal intracapillary and arteriolar thrombi. Drug-dose reduction or withdrawal was effective in renal function preservation, but cancer progressed in all patients. The management of lenvatinib-induced renal TMA remains a challenge. The best therapy in these patients is still uncertain. Earlier and more precise measurement of urine protein levels, allowing for early dose adjustment, could be effective in preventing further damage and drug discontinuation.

Sulforaphane as a Promising Natural Molecule for Cancer Prevention and Treatment.

Tumorigenicity-inhibiting compounds have been identified in our daily diet. For example, isothiocyanates (ITCs) found in cruciferous vegetables were reported to have potent cancer-prevention activities. The best characterized ITC is sulforaphane (SF). SF can simultaneously modulate multiple cellular targets involved in carcinogenesis, including (1) modulating carcinogen-metabolizing enzymes and blocking the action of mutagens; (2) inhibition of cell proliferation and induction of apoptosis; and (3) inhibition of neo-angiogenesis and metastasis. SF targets cancer stem cells through modulation of nuclear factor kappa B (NF-κB), Sonic hedgehog (SHH), epithelial-mesenchymal transition, and Wnt/β-catenin pathways. Conventional chemotherapy/SF combination was tested in several studies and resulted in favorable outcomes. With its favorable toxicological profile, SF is a promising agent in cancer prevention and/or therapy. In this article, we discuss the human metabolism of SF and its effects on cancer prevention, treatment, and targeting cancer stem cells, as well as providing a brief review of recent human clinical trials on SF.

Scar-reducing effects of gambogenic acid on skin wounds in rabbit ears

Hypertrophic scar (HS) is a dermal fibroproliferative disease that often occurs following abnormal wound healing. To date, there is no satisfied treatment strategies for improvement of scar formation with few side effects. The effects of gambogenic acid (GNA) on scar hypertrophy has not been studied previously. The present study was undertaken to find out the scar-reducing effects of GNA (0.48, 0.96 or 1.92 mg/ml) on skin wounds in rabbit ears. Scar evaluation index (SEI), collagen I (Col1) and collagen III (Col3), microvascular density (MVD), CD4+T cells and macrophages, vascular endothelial growth factor receptor 2 (VEGFR2), fibroblast growth factor receptor 1 (FGFR1), phospho-VEGFR 2 (p-VEGFR2) and p-FGFR1, interleukin (IL)-1β, IL-6, IL-10 and tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) in scar tissue were detected using various methods, respectively. Our data showed that GNA significantly reduced SEI, and the expression of Col1 and Col3 in scar tissue in a concentration-dependent manner. Also, it decreased MVD, the infiltration of CD4+T cells and macrophages, and the levels of VEGFR2, p-VEGFR2, FGFR1, p-FGFR1, TGF-β1, CTGF, IL-1β, IL-6, TNF-α, in addition to upregulated IL-10 in scar tissue. As a result, this study revealed that GNA reduced HS formation, which was associated with the inhibition of neoangiogenesis, local inflammatory response and growth factor expression in scar tissue during wound healing. These findings suggested that GNA may be considered as a preventive and therapeutic candidate for HS.

Important role of annexin A2 (ANXA2) in new blood vessel development in vivo and human triple negative breast cancer (TNBC) growth

Development of new blood vessels in the tumor microenvironment is an essential component of tumor progression during which newly formed blood vessels nourish tumor cells and play a critical role in rapid tumor growth, invasion and metastasis. Nevertheless, how tumor cells develop new blood vessels in the tumor microenvironment (TME) have been enigmatic. Previously, we have shown specific overexpression of ANX A2 in TNBC cells regulates plasmin generation and suspected a role in neoangiogenesis. In this report, we used Matrigel plug model of in vivo angiogenesis and confirmed its role in new blood vessel development. Next, we tested if blocking of ANX A2 in aggressive human breast TME can inhibit angiogenesis and tumor growth in vivo. We showed that aggressive human breast tumor cells growing in nude mice can induce intense neoangiogenesis in the tumor mass. Blocking of ANXA2 significantly inhibited neoangiogenesis and resulted in inhibition of tumor growth. Interestingly, we identified that blocking of ANXA2 significantly inhibited tyrosine phosphorylation (Tyr-P) of ANXA2 implying its involvement in tyrosine signaling pathway and suggesting it may regulate angiogenesis. Taken together, our experimental evidence suggests that ANX A2 could be a novel strategy for disruption of tyrosine signaling and inhibition of neoangiogenesis in breast tumor.

Pathogenetic aspects of post-surgical adhesions prevention (review of literature)

Introduction. It has been established that adhesions in the abdominal cavity are a natural protective and adaptive reaction in response to peritoneal damage, but excessive adhesions are not only a pathological process, but also causes intestinal obstruction in children in 62%.Purpose: to study the pathogenetic aspects of prevention of surgical adhesions.Results. A large number of preventive strategies have been proposed, which are aimed at certain adhesion etiopathogenesis zones, such as inflammation and exudation reduction; coagulation cascade inhibition, fibrin matrix and fibrin deposition formation; fibrinolysis process potentiation; fibroblast proliferation inhibition; neoangiogenesis inhibition; free radical oxidation prevention caused by hypoxia; prevention of contact between damaged peritoneal areas.Conclusions. The problem of prevention of excessive intra-abdominal adhesions remains relevant due to imperfection of the existing variety of methods of prevention and treatment of adhesive intestinal obstruction, and one of the main tasks of its solution in children is to find a sparing and, at the same time, effective way to prevent excessive intra-abdominal adhesions at preoperative, intraoperative and postoperative stages.

Abstract 1902: Enrichment of putative cancer stem cells during anti-angiogenic therapies promotes relapse formation in hepatocellular carcinoma

Abstract Background and Aims: Activation of neo-angiogenic processes in hepatocellular carcinoma (HCC) during disease progression is frequently associated with poor clinical outcome. Consequently, inhibition of neo-angiogenesis is an effective treatment strategy for advanced HCC. However, development of chemoresistance is observed in the majority of patients. Compelling evidence suggest that cancer stem cells (CSCs) may contribute to the acquisition of resistant properties in many solid tumors, but their exact role in this process for HCC remains to be defined. Here, we evaluate the importance of CSCs in the development of resistance and relapse formation after exposure to different anti-angiogenic therapies in HCC and define the concomitant adaptive molecular changes. Methods: Five HCC cell lines and one primary HCC isolate were exposed to sorafenib and sunitinib for a total of 14 days. The treatment effects on CSCs were estimated by sphere forming capacity in vitro as well as the side-population (SP) approach. Expression levels of key oncogenic and CSC markers, such as EpCAM, CD133 and ABCG2 transporter, were assessed by qRT-PCR and flow cytometry. Furthermore, whole transcriptome analyses were performed at different time points. Results: Both treatment regimens effectively reduced oncogenic properties in all investigated HCC cells. However, sustained anti-proliferative effect after treatments was observed in only one cell line. In three other hepatoma lines an initial treatment effect was subsequently followed by rapid re-growth thereby mimicking the responses observed in patients. Interestingly, two cell lines showed differential response to applied drugs, showing anti-proliferative effects to sorafenib, while relapse formation occurred after sunitinib treatments. While anti-oncogenic effects in sensitive cell lines were associated with significant reduction in sphere forming capacity, CSC marker EpCAM as well as SP cells, resistant cell line showed a transient increased in CSC properties. Importantly, acquired resistance to both drugs uniformly developed in the cell lines suggesting that common molecular mechanisms might be operative. These adaptive molecular changes involved signaling pathways known to be associated to cell survival and proliferation (RAS, AKT, MYC), as well as angiogenesis (VEGFR, PDGFR, HIF1a). Furthermore, the resistant cell lines showed compensatory upregulation of key oncogenic molecules such as EGFR as well as multidrug resistance ABC transporters. Conclusions: Our in vitro model recapitulates features of drug resistance observed in human HCC patients. Resistance to anti-angiogenic therapies might be fueled by transient expansion of CSCs. Therefore, specific targeting of CSCs as well as pro-oncogenic compensatory signaling pathways might be an effective therapeutic strategy to overcome resistance in HCC. Citation Format: Darko Castven, Carolin Czauderna, Diana Becker, Marcus A. Wörns, Snorri S. Thorgeirsson, Peter Grimminger, Hauke Lang, Peter R. Galle, Jens U. Marquardt. Enrichment of putative cancer stem cells during anti-angiogenic therapies promotes relapse formation in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1902. doi:10.1158/1538-7445.AM2017-1902

Hypericin targets multiple signaling mediators in cancer cells generating unique, diverse anti-tumoral, anti-metastatic, and anti-angiogenic activities with evidence for clinical applicability

The goals of this review are to comprehensively analyze the diverse biological activities displayed by a most potent photodynamic agent–hypericin. Hypericin is a lipophilic redox-reactive molecule possessing a redox potential low enough to act as electron acceptor, subsequently discharging these electrons to oxygen, generating ROS. This property enables intracellular hypericin to retain redox activities in the dark. In cells hypericin sequesters in endoplasmic reticulum and Golgi apparatus membranes and photo-oxidizes membranal lipoproteins. However, the most relevant cytosolic hypericin target is the Hsp90 chaperone. We have shown that hypericin selectively binds to and oxidizes Hsp90, inducing its forced polyubiquitiylation, functional inactivation and rapid degradation in a proteasome independent manner. Hsp90 physiological association with a myriad of client proteins is disrupted and several signaling mediators, cell cycling and proliferation regulators are destabilized and degraded. Secondarily affected cell cycle checkpoints cause uneven, premature mitosis, (karyokinesis with no cytokinesis), forming polykaryonic giant cells, hallmark of mitotic catastrophe also known as mitotic cell death. HIF-1a, the master regulator of VEGF synthesis and angiogenesis inducer is also an Hsp90 client protein. HIF-1a is physiologically degraded by oxygen but also by the hypericin-induced Hsp90 ablation, inducing potent tumor neoangiogenesis inhibition. Hsp90 is implicated in mediating inheritable epigenetic modifications, causing epigenetic signature changes in key developmentally regulated genes and tumor cell exit from proliferation cycles. Expression of EZH2, the Polycomb repressor complex-2 catalytic subunit, which trimethylates histone H3lys27 is suppressed, class-I HDACs expression downregulated and HDAC1-Dnmt1-EZH2 complex formations diminish. Deficiencies in HDACs cellular contents lead to histones H3 and H4 hyperacetylation, which together with diminished H3K27-trimethylation relax chromatin structure, activating transcription including of differentiation-promoting genes. In GBM cells neuroglial differentiation antigens are expressed, cytoarchitecture modulated and the cells undergo tumor cell differentiation. Indeed, clinically significant anti-GBM effects were obtained in a clinical trial in recurrent, progressive GBM patients.

Structural Alteration in Dermal Vessels and Collagen Bundles following Exposure of Skin Wound to Zeolite-Bentonite Compound.

Background. This study examines the impact of one-time direct application of haemostatic agent zeolite–bentonite powder to wounded skin on the healing process in rats. Materials and Methods. 24 male Sprague-Dawley rats were randomly allocated into two groups (n = 12): (1) the rats whose wounds were washed only with sterile normal saline (NS-treated) and (2) those treated with zeolite–bentonite compound (ZEO-treated). The wound was circular, full-thickness, and 2 cm in diameter. At the end of the 12th day, six animals from each group were randomly selected and terminated. The remaining rats were terminated after 21 days. Just after scarification, skin samples were excised and sent for stereological evaluation. Results. The results showed a significant difference between the two groups regarding the length density of the blood vessels and diameter of the large and small vessels on the 12th day after the wound was inflicted. Besides, volume density of both the dermis and collagen bundles was reduced by 25% in the ZEO-treated rats in comparison to the NS-treated animals after 21 days. Conclusions. One-time topical usage of zeolite–bentonite haemostatic powder on an animal skin wound might negatively affect the healing process through vasoconstriction and inhibition of neoangiogenesis.

HIF-1α in Myeloid Cells Promotes Adipose Tissue Remodeling Toward Insulin Resistance.

Adipose tissue hypoxia is an important feature of pathological adipose tissue expansion. Hypoxia-inducible factor-1α (HIF-1α) in adipocytes reportedly induces oxidative stress and fibrosis, rather than neoangiogenesis via vascular endothelial growth factor (VEGF)-A. We previously reported that macrophages in crown-like structures (CLSs) are both hypoxic and inflammatory. In the current study, we examined how macrophage HIF-1α is involved in high-fat diet (HFD)-induced inflammation, neovascularization, hypoxia, and insulin resistance using mice with myeloid cell-specific HIF-1α deletion that were fed an HFD. Myeloid cell-specific HIF-1α gene deletion protected against HFD-induced inflammation, CLS formation, poor vasculature development in the adipose tissue, and systemic insulin resistance. Despite a reduced expression of Vegfa in epididymal white adipose tissue (eWAT), the preadipocytes and endothelial cells of HIF-1α-deficient mice expressed higher levels of angiogenic factors, including Vegfa, Angpt1, Fgf1, and Fgf10 in accordance with preferable eWAT remodeling. Our in vitro study revealed that lipopolysaccharide-treated bone marrow-derived macrophages directly inhibited the expression of angiogenic factors in 3T3-L1 preadipocytes. Thus, macrophage HIF-1α is involved not only in the formation of CLSs, further enhancing the inflammatory responses, but also in the inhibition of neoangiogenesis in preadipocytes. We concluded that these two pathways contribute to the obesity-related physiology of pathological adipose tissue expansion, thus causing systemic insulin resistance.

Tumor-initiating cells contribute to acquired chemoresistance during anti-angiogenic therapies in hepatocellular carcinoma

Inhibition of neo-angiogenesis is an effective treatment strategy for advanced HCC. However, development of chemoresistance is observed in the majority of patients. Compelling evidence suggest that stem-like tumor-initiating cells (TICs) may contribute to the acquisition of resistant properties in many solid tumors, but their exact role in this process for HCC remains to be defined. Here, we evaluate the importance of TICs in the development of resistance to different anti-angiogenic therapies in HCC and define concomitant adaptive molecular changes. Several HCC cell lines and primary HCC isolates were exposed to sorafenib and sunitinib for total of 14 – 21 days. Treatment effects on TICs were estimated by sphere forming capacity and side-population (SP) approach. Expression levels of key oncogenic and TIC markers were assessed by qRT-PCR and flow cytometry. Whole transcriptome analyses were performed at different time points. Both treatments effectively reduced oncogenic properties in all investigated HCC cells. Sustained anti-proliferative effects were observed in only two cell lines whereas an initial treatment effect was subsequently followed by rapid re-growth in the majority of HCC cells thereby mimicking responses observed in patients. While anti-oncogenic effects in sensitive cell lines were associated with significant reduction sphere forming capacity, TIC markers as well as SP cells, resistant cell line showed transient increased in TIC properties. Acquired resistance to both drugs uniformly developed in the cell lines suggesting that common molecular mechanisms might be operative. These adaptive molecular changes involved signaling pathways known to be associated to cell survival (ERK, AKT, MYC), proliferation (TP53, CDKN1A) and angiogenesis (VEGF, PDGFR, HIF1a). The resistant cell lines showed compensatory upregulation of key oncogenic molecules such as EGFR as well as multidrug resistance ABC transporters. Our in vitro model recapitulates features of drug resistance observed in human HCC patients. Resistance to anti-angiogenic therapies might be fueled by transient expansion of TICs. Therefore, specific targeting of TICs as well as pro-oncogenic compensatory signaling pathways might be an effective therapeutic strategy to overcome resistance in HCC.

Acute and sub-chronic toxicity studies of three plants used in Cameroonian ethnoveterinary medicine: Aloe vera (L.) Burm. f. (Xanthorrhoeaceae) leaves, Carica papaya L. (Caricaceae) seeds or leaves, and Mimosa pudica L. (Fabaceae) leaves in Kabir chicks

Ethnopharmacological relevanceAloe vera (L.) Burm. f. (Xanthorrhoeaceae), Carica papaya L. (Caricaceae) and Mimosa pudica L. (Fabaceae) are widely used in the Cameroonian ethnoveterinary medicine as a panacea, and specifically for gastrointestinal disorders as well as an anthelmintic and antibacterial. Aim of the studyThe present study evaluated the potential toxicity of the hydroalcoholic extracts of Aloe vera leaves, Carica papaya leaves or seeds, and Mimosa pudica leaves after acute and sub-chronic administration in chicks. Materials and methodsFor the acute toxicity test a single administration of each of the four hydroalcoholic extracts was given orally at doses ranging from 40 to 5120mg/kg (n=5/group/sex). In the sub-chronic study, these extracts were given orally as a single administration to chicks at doses of 80, 160, 320 and 640mg/kg/day for 42 days. The anti-angiogenic properties of these extracts (5–320µg/mg) were investigated in the chick chorioallantoic membrane in vivo. ResultsIn the acute toxicity test, none of the four studied hydroalcoholic extracts induced mortality or significant behavioural changes. The sub-acute treatment with the four plant extracts did not alter either the body weight gain or the food and water consumption. However, the results indicated that Aloe vera leaf extract acute treatment by oral route at doses up to 2560mg/kg did not produce death in 50% (5/10) of chicks during 24h or 14 days of observation, but 20% (2/10) chicks died. The haematological and biochemical analyses did not show significant differences in any of the parameters examined in female or male groups, with the exception of a transient rise in white blood cell counts at high doses (640mg/kg). Additionally, these extracts did not have the potential for anti-angiogenic effects through the inhibition of neo-angiogenesis in the chick chorioallantoic membrane in vivo. ConclusionThe results showed that the therapeutic use of the hydroalcoholic extracts of Aloe vera leaves, Carica papaya leaves or seeds and Mimosa pudica leaves had very low toxicity in oral acute high dose administration and no toxicity in oral sub-chronic low dose administration and indicate that the plants could be considered safe for oral medication in chicks.

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

Leucas aspera inhibits the Dalton's ascitic lymphoma in Swiss albino mice: A preliminary study exploring possible mechanism of action.

Background:North East India is a rich source of medicinal plants and a number of plant extracts are used by tribal peoples living in this area for various disorders. L.aspera is such a plant, traditionally used as an antitumor agent.Aim:In the present study, aerial parts of L.aspera were investigated for antitumor activity in Dalton's lymphoma (DAL) bearing mice. The ability of plant extract in free radical scavenging, neoangiogenesis inhibition and macrophage stimulation were also checked.Materials and Methods:Based on the preliminary in vitro cytotoxicity studies ethyl acetate fraction of L.aspera (EALA) was selected for the detailed study. DAL ascites tumor model was performed to check the antitumor activity of EALA (200 and 400mg/kg of body weight). Hematological and histopathological parameters were estimated. Antioxidant levels, neoangiogenesis and peritoneal macrophage count were also determined.Results:In vitro MTT and Trypan blue assay results showed the cytotoxic effect of EALA in DAL cells lines. EALA treatment resulted in significant decrease in ascites tumor volume and viable cell count. Hematological and liver antioxidant parameters were normalised by EALA treatment. It was also found that EALA treatment inhibits neovascularisation and produce macrophage stimulation in treated mice.Conclusion:The results showed that EALA is a promising anticancer agent and its activity is comparable to the standard drug 5-Flouro uracil (5-FU).

Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell ApoptosisIn VivoPrimarily by Inhibiting VEGF Expression and Angiogenesis

We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

The Internal Ribosome Entry Site-Medaited Expression of VEGF Rescues HS Sultan Tumor Cells From Mammalian Target of Rapamycin (mTOR)-Inhibitors Induced Apoptosis in Vivo

Abstract 1659An important molecular target of mTOR inhibitors in cancer therapy is VEGF expression and neo-angiogenesis. In prior studies, we demonstrated that, although rapalog mTOR inhibitors only induce G1 arrest in B-cell tumor lines, their administration in vivo in xenograft models resulted in tumor cell apoptosis that correlated with inhibition of neo-angiogenesis and VEGF expression within the tumor bed. Other in vitro studies have shown that IRES-dependent translation of myc and D-cyclins can provide a fail-safe mechanism for expression when mTOR inhibitors prevent cap-dependent translation and may act as a resistance mechanism to induction of G1 arrest in vitro. We, thus, tested if VEGF IRES activity could likewise regulate induction of anti-tumor effects and apoptosis in vivo. To test if VEGF IRES activity can regulate anti-tumor responses in vivo, we utilized the HS-Sultan B cell lymphoma line that is PTEN null. Its heightened AKT activity disarms the VEGF IRES, preventing this fail-safe mechanism and sensitizing to mTOR inhibitors. We ectopically expressed a version of the VEGF ORF in these cells fused to the p27 IRES (p27-VEGF), an IRES which is insensitive to AKT and effective in PTEN-null tumors. p27-VEGF transfected tumor cells were used as subcutaneous challenges in immunodeficient mice and results of mTOR inhibitor treatment compared to control mice challenged with tumor cells transfected with the VEGF ORF but without an AKT-resistant IRES. The anti-tumor responses were enumerated by assessing tumor size and tumor apoptosis, neo-angiogenesis and VEGF expression assessed by immunohistochemistry, ELISA, and Western blot analysis of tumor lysate. Ectopic expression of VEGF fused to the p27 IRES specifically enhanced VEGF expression and neo-angiogenesis in tumors of mice treated with the rapalog temsirolimus or active site mTOR inhibitor pp242 and significantly reduced tumor cell apoptosis and anti-tumor responses. The results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and, furthermore, underscore the importance of IRES activity as a resistance mechanism to such targeted therapy. Disclosures:No relevant conflicts of interest to declare.